1. Field of the Invention
The present invention relates to a method of altering an enzyme, in particular, to a method of altering an enzyme to enhance its transfer reaction, and to a novel neopullulanase altered using said method.
2. Description of the Prior Art
Conventional methods used to enhance enzymatic transfer reactions are to change the external environment for enzyme reaction, for example, an enzyme reaction is carried out, not in an aqueous solution, but in an organic solvent. The reaction mechanism of a hydrolysis reaction and a transfer reaction are the same, in principle, in terms of an organic chemical reaction; a reaction is called hydrolysis when the acceptor is a water molecule, whereas it is called a transfer reaction when the acceptor is a compound other than water. Many transferases have catalytic activities for both hydrolytic and transfer reactions. Thus, the transfer reaction of such transferases can be enhanced by carrying out the enzyme reaction in an organic solvent so as to suppress the hydrolytic reaction.
In contrast, in order to enhance the transfer reaction an aqueous solution, it is necessary to alter an enzyme itself to increase its indigenous transfer activity. Genetic engineering and protein engineering are basic technologies to alter an enzymes itself to improve its characteristics for industrial use (e.g., stability, substrate specificity and reaction specificity). An example of the use of these technologies to improve the stability of a protein is described in Japanese Patent Laid-Open No. 224489/1985 in which additional cysteine residues are introduced into a native protein; these are linked via a disulfide bond, which increased the stability of the protein. In this connection, a method has been developed to determine a site to introduce the disulfide bonding using computer modeling (U.S. Pat. No. 4,853,871). Moreover, several other techniques to improve stability of proteins have been disclosed. Further, an example of altering the substrate specificity of an enzyme was given by some members of the present invention, in which they proposed alteration of the substrate specificity using protein engineering to obtain a novel dairy lactic acid bacterium protease having a substrate decomposing activity entirely different from that of the corresponding wild-type enzyme (Japanese Patent Application No. 190119/1992; Laid-Open No. 153945/1994). A novel protease having a specific activity higher than a corresponding natural-type enzyme was also disclosed in this Patent Application. Also, in Japanese Patent Laid-Open No. 20291/1992, some members of the present invention disclosed a neopullulanase in which its substrate specificity was altered by protein engineering technology.
It is known that the neopullulanase, used in the present invention, derived from Bacillus stearothermophilus, acts on polysaccharides and oligosaccharides, such as starch and pullulan, hydrolyzes the alpha-1,4- and alpha-1,6- glucosidic bonds, and forms alpha-1,4- and alpha-1,6- glucosidic bonds by sugar transfer reactions (J. Biol. Chem., 267, 18447-18452, 1992).
Of the conventional methods to enhance enzymatic transfer reactions, the method in which the enzyme reaction proceeds in an organic solvent cannot be applied to the food industry because of the toxicity of the organic solvent. Increased manufacturing costs by the use of an organic solvent is another problem.
On the other hand, of the conventional methods mentioned above, the method of altering the enzyme itself using protein engineering technology solves the problems of the toxicity and cost. However, although means for altering the stability and substrate specificity of natural-type enzymes are known as described above, concrete means for altering reaction specificities so as to enhance transfer reactions are not known. This is because problems remain unsolved with regard to the structural stability of enzyme themselves or enzyme-substrate complexes, and the mechanisms of enzymatic reactions and relevant factors thereof must be better understood. Furthermore, it seems not only extremely inefficient, but actually impossible to alter enzymes so as to control their reactivity using a trial-and-error substitution method based mainly on information about amino acid sequences, since function and physicochemical characteristics of enzymes are closely related to their three-dimensional structures.